The pancreas is an exocrine gland that secretes digestive enzymes directly into the digestive tract as well as an endocrine gland that secretes hormones into the blood stream. The exocrine function is assured by acinar and centroacinar cells that secrete various digestive enzymes via intercalated ducts into the duodenum. The functional unit of the endocrine pancreas is the islet of Langerhans. Islets are scattered throughout the exocrine portion of the pancreas and are composed of four main cell types: alpha-, beta-, delta- and PP-cells (reviewed for example in Kim & Hebrok, 2001, Genes Dev. 15: 111-127). Beta-cells produce insulin, represent the majority of the endocrine cells and form the core of the islets, while alpha-cells secrete glucagon and are located in the periphery. Delta-cells and PP-cells are less numerous and secrete somatostatin and pancreatic polypeptide, respectively. Recently, cells producing the neuropeptide Ghrelin have been found in pancreatic islets (Wierup et al., 2002, Regal Pept. 107:63-9.).
Early pancreatic development has been well studied in different species, including chicken, zebraflsh, and mice (for a detailed review, see Kim & Hebrok, 2001, supra). The pancreas develops from distinct dorsal and ventral anlagen. Pancreas development requires specification of the pancreas anlage along both anterior-posterior and dorsal-ventral axes. A number of transcription factors, that are critical for proper pancreatic development have so been identified (see Kim & Hebrok, 2001, supra; Wilson et al., 2003, Mech Dev, 120: 65-80).
In humans, the acinar and ductal cells retain a significant proliferative capacity that can ensure cell renewal and growth, whereas the islet cells become mostly mitotically inactive. This is in contrast to rodents where beta-cell replication is an important mechanism in the generation of new beta cells. It has been suggested, that during embryonic development, pancreatic islets of Langerhans originate from differentiating duct cells or other cells with epithelial morphology (Bonner-Weir & Shama, 2002, J Pathol. 197: 519-526; Cu et al., 2003, Mach Day. 120: 35-43). In adult humans, new beta-cells arise in the vicinity of ducts (Butler at al., 2003, Diabetes 52: 102-110; Bouwens & Pipeleers 1998, Diabetologia 41: 629-633). However, also an intra-islet location or an origin in the bone marrow has been suggested for precursor cells of adult beta-cells (Zulewski et al., 2001, Diabetes 50: 521-533; lanus at al., 2003, J Clin Invest. 111: 843-850). Pancreatic islet growth is dynamic and responds to changes in insulin demand, for example during pregnancy or due to changing body weight during childhood. In adults, there is a good correlation between body mass and islet mass (Yoon et al., 2003, J Clin Endocrinol Metab. 88: 2300-2308).
Pancreatic beta-cells secrete insulin in response to blood glucose levels. Insulin amongst other hormones plays a key role in the regulation of the fuel metabolism. Insulin leads to the storage of glycogen and triglycerides and to the synthesis of proteins. The entry of glucose into muscles and adipose cells is stimulated by insulin. In patients who suffer from diabetes mellitus type I or LADA (latent autoimmune diabetes in adults (Pozzilli & Di Mario, 2001, Diabetes Care. 8:1460-67) beta-cells are being destroyed due to autoimmune attack. The amount of insulin produced by the remaining pancreatic islet cells is too low, resulting in elevated blood glucose levels (hyperglycemia). In diabetes type II liver and muscle cells loose their ability to respond to normal blood insulin levels (insulin resistance). High blood glucose levels (and also high blood lipid levels) in turn lead to an impairment of beta-cell function and to an increase in beta-cell apoptosis. It is interesting to note that the rate of beta-cell neogenesis does not appear to change in type II diabetics (Butler et al., 2003 supra), thus causing a reduction in total beta-cell mass over time. Eventually the application of exogenous insulin becomes necessary in type II diabetics.
Improving metabolic parameters such as blood sugar and blood lipid levels (e.g. through dietary changes, exercise, medication or combinations thereof) before beta-cell mass has fallen below a critical threshold leads to a relatively rapid restoration of beta-cell function. However, after such a treatment the pancreatic endocrine function would remain impaired due to the only slightly increased regeneration rate.
In type I diabetics, where beta-cells are being destroyed by autoimmune attack, treatments have been devised which modulate the immune system and may be able to stop or strongly reduce islet destruction (Raz et al., 2001, Lancet 3513: 1749-1753; Chatenoud et al., 2003, Nat Rev Immunol. 3: 123-132; Homann et al., Immunity. 2002, 3:403-15). However, due to the relatively slow regeneration of human beta-cells such treatments can only be successful if they are combined with agents that can stimulate beta-cell regeneration.
Diabetes is a very disabling disease, because today's common anti-diabetic drugs do not control blood sugar levels well enough to completely prevent the occurrence of high and low blood sugar levels. Out of range blood sugar levels are toxic and cause long-term complications like for example renopathy, retinopathy, neuropathy and peripheral vascular disease. There is also a host of related conditions, such as obesity, hypertension, heart disease and hyperlipidemia, for which persons with diabetes are substantially at risk.
Apart from the impaired quality of life for the patients, the treatment of diabetes and its long term complications presents an enormous financial burden to our healthcare systems with rising tendency. Thus, for the treatment of, type I and type II diabetes as well as for latent autoimmune diabetes in adults (LADA) there is a strong need in the art to identify factors that induce regeneration of pancreatic insulin producing beta-cells. These factors could restore normal function of the endocrine pancreas once its function is impaired or event could prevent the development or progression of diabetes type I, diabetes type II, or LADA.
In this invention, we disclose a novel and so far unknown use for the neurotrophic factor neurturin to stimulate the formation or regeneration of insulin producing beta-cells and thus, a use in the treatment and prevention of diabetes.
Neurotrophic factors are growth factors that regulate the survival and maintain the phenotypic differentiation of certain nerve and/or glial cell populations (Varon at al., 1978, Ann. Rev. Neurosience 1: 327-361; Thoenen at al., Science, 229:238-242, 1985). Nerve growth factor (NGF) was the first neurotrophic factor to be identified and characterized (Levi-Montalcini at al., 1951, J. Exp. Zool. 116:321). The second member of this family to be discovered was brain-derived neurotrophic factor (Leibrock at al., 1989, Nature 341:149-152).
Glial-derived neurotrophic factor (GDNF)® a neurotrophic factor structurally unrelated to NGF—was discovered during a search for factors crucial to the survival of midbrain dopaminergic neurons, which degenerate in Parkinson's disease (Lin at al., 1993, Science 260:1130-2). Sequence analysis revealed it to be a distant member of the superfamily of transforming growth factor 8 (TGF-beta) factors.
Another neurotrophic factor that is structurally closely related to GDNF and unrelated to NGF is neurturin (Kotzbauer et al., 1996, Nature 384: 467-470). Neurturin, GDNF and two other related factors (artemin and persephin) define a family of neurotrophic factors referred to as TGF-beta-related neurotrophins. These neurotrophic factors promote the survival of various neurons including peripheral autonomic and sensory neurons as well as central motor and dopamine neurons, and have been proposed as therapeutic agents for neurodegenerative diseases (see review by Takahashi, 2001, Cytokine Growth Factor Rev 12(4):361-73; see also, for example, U.S. Pat. No. 6,090,778 and EP1005358B1, the disclosures of which are hereby incorporated by reference). TGF-beta-related neurotrophins signal through a unique two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha (GFRalpha) and receptor protein tyrosine kinase Ret.
Apart from the described functions in neuronal tissue GDNF/RET signalling is crucial for the differentiation of certain non-neuronal tissues. For example, GFRalpha1 and the Ret are both necessary receptor components for ureteric bud outgrowth and subsequent branching in the developing kidney (Cacalano at al., 1998, Neuron 21:53-62; Tang at al., 1998, J. Cell Biol. 142 (5):1337-45). Ret, GFRalpha-1 (the GDNF receptor), and GFRalpha-2 (the Neurturin receptor) are expressed by testicular germ cells, while GDNF and Neurturin are expressed by Sertoli cells. Both GDNF and Neurturin stimulate DNA synthesis in spermatogonia. Furthermore, GFRalpha, GFRalpha ligands and co-receptors are expressed in germ cell tumors and thus may act as paracrine factors in spermatogenesis (Viglietto at al., 2000, Int J Oncol. 16(4):689-94).
Only recently, it was shown that the biology of GDNF signalling is much more complex than originally assumed. GDNF family ligands also signal through the neural cell adhesion molecule NCAM. In cells lacking Ret, GDNF binds with high affinity to the NCAM and GFRalpha1 complex (see review by Sariola & Saarma, 2003, J Cell Sol. 116(Pt 19):3855-62). Signalling via the c-met receptor kinases has also been demonstrated (see Popsueva et al., 2003, J Cell Biol. 161(1):119-29).
Although it has been discussed in the prior art that GDNF/RET signalling is crucial for the differentiation of neuronal and certain non-neuronal tissues, it has not been disclosed that a member of family of TGF-beta-related neurotrophins is involved in the regeneration of pancreatic tissue. We found surprisingly, that neurturin stimulates the formation or regeneration of insulin producing pancreatic beta-cells which play an essential role in diabetes. Thus, in this invention, we disclose the use of neurturin in the treatment and prevention of diabetes.